The long-term objective of this research program is to understand the molecular mechanisms involved in the regulation of intracellular cholesterol metabolism. Based on results presented in PRELIMINARY STUDIES, in the forthcoming granting period the focus will be on studying the mechanisms of cellular cholesterol trafficking and storage processes in mammalian cells. Two Specific Aims are proposed: Specific Aim 1: To test the hypothesis that the trans-Golgi network (TGN) serves as a key organelle for transporting low-density lipoprotein (LDL)-derived cholesterol from the endosomes to the plasma membrane/endoplasmic reticulum (ER). SubAim 1.1: To test the possibility that membranes containing the Syntaxin 6/Syntaxin 16/Vti1a complex play a key role in LDL-CHOL trafficking. SubAim 1.2: To test the possibility that the v-SNAREs Vamp4/Vamp3 may contribute to LDL- CHOL transport between the NPC1 compartment and the Syntaxin 6-rich compartment. SubAim 1.3: To biochemically characterize various membrane compartments associated with 3H-CHOL derived from LDL. Specific Aim 2: To examine the functional and biochemical characteristics of caveolae in the ER. SubAim 2.1: To examine the functional characteristics of the ER caveolae in intact cells and in vitro. SubAim 2.2: To examine the biochemical characteristics of the ER caveolae in intact cells and in vitro. LDL is the major cholesterol carrier in the blood. High LDL is a risk factor for atherosclerosis, which is a major cause of heart disease. The outcome of research described in the current application will bring new knowledge about the intracellular transport and storage processes of LDL-derived cholesterol, and may help develop new approaches for reducing the amount of LDL-derived cholesterol stored in tissues. PUBLIC HEALTH RELEVANCE: Low-density lipoprotein (LDL) is the major cholesterol carrier in the blood. High LDL is a risk factor for atherosclerosis, which is a major cause of heart disease. The outcome of research described in the current application will bring new knowledge about the transport and storage process of LDL-derived cholesterol, and may help develop new approaches for reducing the amount of LDL-derived cholesterol stored in tissues.